The long-term goal of this project is to identify the cellular and molecular mechanisms of Human Cytomegalovirus (HCMV) persistence in the host. Studies by our group and others have implicated endothelial cells and macrophages as potential reservoirs of the virus in the host. In the previous funding period we have shown that HCMV can persistently infect aortic endothelial cells (EC) in culture. We have also demonstrated HCMV sero-positive asymptomatic individuals harbor latent HCMV in peripheral blood myeloid cells, and that this virus can be reactivated and isolated from allogeneically-stimulated monocyte derived macrophages (MDM). These studies from our laboratory and observations of other investigators have shown that MDM and EC play an important, if not fundamental role in the biology and pathogenesis of HCMV. However, our understanding of HCMV replication and persistence in these cell types is limited, primarily due to inconsistent and restricted infection of MDM and EC by genetically characterized laboratory strains of HCMV. Importantly, several groups, including our own have observed that the type of HCMV strain used to infect MDM and EC is critical for efficient infection. We have recently characterized a number of low passage clinical HCMV isolates for their growth in MDM and EC and have identified a strain (TR), which can consistently infect MDM as well as EC with high efficiency (100%). We hypothesize that HCMV encodes one or more genetic elements that enables the virus to infect and replicate in MDM and EC. Therefore, our goals in this project are to identify the viral genes responsible for these viral phenotypes and to characterize the mechanisms and products encoded by these genes that promote growth in MDM and EC. To address this objective, the first part of this project will focus on the identification of the HCMV genes that determine growth in MDM and EC by first examining the transcriptional programs of MDM and EC tropic viruses using a HCMV microarray chip to identify potential genes expressed by the tropic but not the non-tropic viruses. Coordinated with this effort we will also generate recombinant viruses between MDM or EC tropic and non-tropic viruses to identify gain and loss of function as well as a transposon library of MDM and EC tropic viruses to identify individual gene(s) involved in this processes. In the final part of this project, we will introduce the MDM and EC tropic genes back into the non-tropic viruses to determine if the genes confer viral growth in MDM and EC. Understanding the genetic basis of viral replication in MDM and EC will further our understanding of virus-host cell interactions that lead to viral persistence in the cell.